MITOGENIC SIGNALING THROUGH RAL A AND PHOSPHOLIPASE D

本建议旨在了解 RalA/磷脂酶D（PLD）信号通路在促有丝分裂信号转导中起重要作用。的 申请人的实验室已经证明PLD活性在细胞中升高 用各种促有丝分裂刺激物处理，并且该途径是一种 v-Src、v-Raf、v-Ras和过表达转化的关键介质 EGF受体在正常静止期细胞中，PLD与GDP结合 RalA和定位于细胞膜。这种酶被激活， RalA GTP交换和Arf GTP向复合物的募集，这两种事件 被认为是由Ras的激活引起的。然而，Arf GTP不 直接结合RalA/PLD复合物，并在细胞膜中升高PLD活性。 Ras转化的细胞需要加入胞质溶胶。这些发现表明 PLD的完全激活需要额外的因素。PLD 将磷脂酰胆碱水解为磷脂酸（PA）。PA参与 许多生物过程，包括细胞内囊泡的形成， 信号分子的激活，但它的确切作用是促进有丝分裂 信令不被理解。申请人实验室的初步证据 表明PLD可能在EGF受体的内吞作用中起作用。两个目标是 建议进一步研究PLD激活机制及其 内吞作用中的假定作用。第一个目的是纯化和表征一个 最近发现的低分子量PLD-刺激因子（PLD-SF）， 在转化和分裂的细胞中升高。第二个目标将考验 PLD通过促进细胞内信号传导调节细胞内信号传导的假说 受体介导的内吞作用产生“信号囊泡"。"

This proposal seeks to understand the role that the RalA/phospholipase D (PLD) signaling pathway plays in mitogenic signaling. The applicant's laboratory has demonstrated that PLD activity is elevated in cells treated with a variety of mitogenic stimuli, and that this pathway is a critical mediator of transformation by v-Src, v-Raf, v-Ras, and overexpression of the EGF receptor. In normal quiescent cells, PLD is complexed with GDP-bound RalA and localized to cellular membranes. The enzyme becomes activated upon RalA GTP exchange and recruitment of Arf GTP to the complex, both events thought to be brought about by activation of Ras. However, Arf GTP does not bind the RalA/PLD complex directly, and elevated PLD activity in membranes of Ras-transformed cells requires the addition of cytosol. These findings suggest that an additional factor is needed for complete activation of PLD. PLD hydrolyzes phosphatidylcholine to phosphatidic acid (PA). PA is involved in many biological processes, including intracellular vesicle formation and activation of signaling molecules, but its exact role in promoting mitogenic signaling is not understood. Preliminary evidence from the applicant's lab suggests that PLD may play a role in EGF receptor endocytosis. Two aims are proposed to further investigate both the mechanism of PLD activation and its putative role in endocytosis. The first aim is to purify and characterize a recently discovered low molecular weight PLD-stimulating factor (PLD-SF) that is elevated in transformed and dividing cells. The second aim will test the hypothesis that PLD regulates intracellular signaling by facilitating receptor-mediated endocytosis to generate "signaling vesicles."